The development of a vertebrate organism from a single fertilized egg is a complex series of events involving both cell proliferation and specialization. Many of these events have been shown to be mediated by intercellular signals. One such family of signaling factors is the Wnt gene family, which is comprised of at least 20 related vertebrate genes (Adamson et el., 1994; Bouillet et al., 1996; Christian et al., 1991; Christiansen et al., 1995; Gavin et al., 1990; Hume and Dodd, 1993; Kispert et al., 1996; Ku and Melton, 1993; Nusse and Varmus, 1982; Roelink and Nusse, 1991; Roelink et al., 1990; Sidow, 1992; Tanda et el., 1995; Wainwright et al., 1988; Wolda and Moon, 1992). Wnt genes encode cysteine- rick polypeptides possessing 50-60% amino acid identify which are though to be secreted signaling molecules involved in cell growth, survival, and, or, differentiation (McMahon et al., 1992; Nusse and Varmus, 1992). My laboratory is particularly interested in the role(s) of directing pluripotential chick somite cells to become skeletal muscle. Although several Wnt family members have been shown to be sufficient and necessary for the induction of myogenesis in vertebrates (Leyns et al., 1997; Munsterberg et el., 1995; Stern et al., 1995; Wang et al., 1997), we are lacking important information about how Wnt proteins act at a cellular level. Despite the identification of the Wnt-1 cDNA more than thirteen years ago (Fung et al., 1985), studies pertaining to the cellular roles of Wnt proteins have been severely hampered by the lack of soluble active ligand. For reasons that are poorly understood, Wnt genes are extremely refractory to over-expression. In this grant, I propose to utilize a COS cell expression system to generate WNT fusion proteins which can easily be purified and used as immunogens for the generation of anti-Wnt monoclonal antibodies. By comparing the biochemical properties of recombinant Wnt proteins and endogenous Wnt proteins, we hope to identify the differences that will help us pinpoint the reason it has been so difficult to generate active soluble recombinant Wnt protein. Furthermore, Wnt protein purified from the COS cell expression system and/or from chick embryos (using immunoaffinity columns) will be used to directly assess the role of Wnt proteins in myogenic specifications. The development of these key reagents will open the door to numerous future studies pertaining to both development and cancer. Another goal of this project is to give students (with particular emphasis on under-represented minorities) an opportunity to do "hands-on" research. The intellectual and technical aspects of this project are quite accessible to both undergraduate and graduate students. This project will provide students with an excellent opportunity to utilize the techniques of biochemistry, molecular biology, cell biology, and developmental biology to address a scientific problem. Students will also be trained to keep good lab notebooks, critically analyze their data, orally present their data, prepare posters for meetings, and prepare manuscripts.